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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. Distribution and Phenotype of Epstein-Barr Virus-Harboring Cells in Hodgkin’s Disease By Hermann Herbst, Erik Steinbrecher, Gerald Niedobitek, Lawrence S. Young, Louise Brooks, Nikolaus Muller-Lantzsch, and Harald Stein Distribution and phenotype of Epstein-Barr virus (EBV)harboring cells were determined in Hodgkin‘s disease (HD) biopsies by in situ hybridization with [35S]-labeled RNA probes specific for the small EBV-encoded nuclear RNAs, EBERl and EBER2, in some instances preceded by immunohistology for CD20, CD30, CD45RO. and CD68 antigens, the T-cell receptor pchain, and latent membrane antigen (LMP) of EBV. Twenty-three of 46 HD cases displayed EBER transcripts in all Hodgkinand Reed-Sternberg (H-RS) cells, and 18 of these cases showed LMP expression exclusively in neoplastic cells. EBER+ small reactive cells were present in 39 cases in low numbers, and in three cases in abundance. Thus, presence of H-RS cells with or without LMP expression was not accompanied by an unrestricted proliferation of reactive EBER+/LMP- lymphoid cells in the majority of HD patients. Simultaneousin situ hybridization with [35S]-labeledimmunoglobulin light chain (IgLC) gene probes and nonisotopically labeled EBER probe showed a phenotype of mature B lymphocytes and a polyclonal composition for a large proportion of the EBER+ small cells. However, in contrast to noninfected cells, CD20 expression was not detectable in many of these cells, which may indicate downregulationof certain differentiation antigens in latently EBV-infected small lymphoid cells in vivo. o 1992 by The American Society of Hematology. HE EPSTEIN-BARR VIRUS (EBV) had been associ- cases, r e ~ p e c t i v e l y .Others ~ ~ , ~ ~ pointed out that most of the EBV genomes detected in tissue extracts could have been derived from EBV-carrying cells of the reactive admixt ~ r e . l More ~-~~ than 90% of adults worldwide are latently infected with this herpesvirus, so that at least some latently infected lymphocytes are likely to be present in almost any lymphoid tissue.22 It was thus argued that the sensitivity limits of in situ hybridization did not permit detection of EBV DNA in the cellular admixture,14raising the possibility that, although responsible for the frequent detection of EBV in HD by PCR, an abundance of EBV-infected cells remained undetected by in situ hybridization for EBV DNA. Detection of the small EBV-encoded nonpolyadenylated RNA transcripts, EBERl and EBER2, by in situ hybridization now provides a way to clarify this issue. This approach has previously been applied to various EBV-associated lesions, such as undifferentiated nasopharyngeal carcioral hairy l e ~ k o p l a k i a CD30+ ,~~ anaplastic largecell lymphoma,25and to a limited number of H D case^.^^.^' The EBERs appear to be transcribed in most human lesions associated with latent EBV infection.28By virtue of their extreme abundance, estimated to be up to lo6 copies per viral genome?9 EBERs lend themselves to the application of radioactive and nonradioactive in situ hybridization, even in routinely formalin-fixed and paraffin-embedded t i s ~ u e s . ~For ~ - ~the 7 present study, we used in sitti hybridization with EBER-specific single-stranded [35S]-labeledantisense and sense (control) RNA probes to address the questions as to the presence, number, and distribution of EBER+ neoplastic and reactive cells in HD tissue biopsies. In bome cases, we additionally used sequential labeling for differentiation antigens by immunohistology and EBER by in situ hybridization, as well as simultaneous in situ hybridization for immunoglobulin light chain (IgLC) RNA transcripts with [35S]-labeled RNA probes and EBER with nonisotopically labeled RNA probes to characterize the phenotype of malignant and reactive EBER+ cells. T ated with Hodgkin’s disease (HD) by various lines of serological and epidemiological e ~ i d e n c e l -prior ~ to the demonstration of EBV antigens and DNA in isolated cases of HD in 1985 and 1987, re~pectively.~?~ Since then, many investigators found EBV genomes in up to 25% of DNA extracts from H D tissue biopsies analyzed by direct filter hybridization methods.6-10Because of the limited sensitivity of this approach, these studies were extended by the application of the polymerase chain reaction (PCR) technique, which showed DNA sequences in considerably larger proportions of cases, ranging between 40% and 80% depending on the sensitivity limits applied.1i-18Although most investigators found approximately 55% to 60% of the H D cases to contain substantial amounts of EBV DNA, the significance of these findings remained controversial. Several investigators favored tumor cells, rather than cells of the reactive admixture, as the most probable origin of the EBV DNA, and this view was substantiated by the visualization of EBV DNA8-11,i9,20 and of the EBV-encoded latent membrane protein (LMP) exclusively in Hodgkin and Reed-Sternberg (H-RS) cells in up to 40% and 48% of H D From the Institute of Patholoo, Klinikum Steglitz, Free University of Berlin, Berlin, Germany; the Department of Cancer Studies, University of Birmingham, Birmingham, UK; and the Division of Kroloo, Institute of Medical Microbiology and Hygiene, University Hospital, University of Saarland, HomburglSaar, Germany. Submitted December 2,1991;accepted March 23,1992. Supported by grants from the Bundesministerium fur Forschung und Technologie (Grant No. 01GA8501), Deutsche Krebshilfe (Grunt No. W91891St2),Deutsche Forschungsgemeinschafr (Grant No. Mu452l2I ) , and the CancerResearch Campaign (to L.S.Y). This work ispartial fulfilment of M.D. thesis requirements (E.S.). Address reprint requests to Hermann Herbst, MD, Institute of Patholoo, Klinikum Steglitz, Free University Berkh, 1 OOO Berlin 45, Germany. The publication costs of this article were defruyed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section I734 solely to indicate this fact. 0 I992 by The American Sociery of Hematology. aoo6-4971/92/8OO2-OO19$3.OO/0 484 MATERIALS AND METHODS Tissues. Lymph node biopsy specimens from 46 HD patients submitted for histopathological diagnosis were routinely processed Blood, Vol80, No 2 (July 15). 1992: pp 484-491 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. EBV IN HODGKIN'S DISEASE by formalin fixation and paraffin embedding. In six cases, snapfrozen aliquots of the tissues, stored at -8O"C, were available. Development of the disease was unrelated to human immunodeficiency virus (HIV) infection, and none of these cases was incorporated into previous studies. Histological typing followed the Rye classification. Ten lymph nodes, removed during mastectomy or prostatectomy and showing a regular histology with only moderate sinus histiocytosis,were used as controls. Immunohistology. The production and specificity profiles of the monoclonal antibodies specific for LMP (clones CS1, -2, -3, and -4)% and of a rabbit antiserum directed against LMP?I are described elsewhere. Monoclonal antibodies against CD20 (L26), CD30 (Ber-Hr2), and CD68 (KP-l), as well as CD3-specificrabbit antibodies, were obtained from Dakopatts (Glostrup, Denmark). The anti-T-cell receptor P-chain antibody, PF1, was from T-cell Sciences (Cambridge, MA), and the CD45RO-specific antibody A633was a kind gift of Dr G.G. Aversa (Stanford, CA). Sections (4 km) of either formol-fixed/paraffin-embedded or snap-frozen tissue blocks were stained by the immunoalkaline phosphatase (APAAP)method." Affinity-purifiedmouse anti-rabbit immunoglobulin, rabbit anti-mouse immunoglobulin antibodies, and APAAP complex (diluted 1:20) were obtained from Dakopatts. Incubation with the CD3-specific antibodies was performed overnight. Formol-fixed sectionsrequired a proteolytic treatment with 1 mg/mL Streptomyces giseus protease (Sigma, Deisenhofen, Germany) for 10 minutes before incubation with the monoclonal antibodies PF1 and Ber-H2. Probes. EBER1- and EBERZspecific fragments were derived from the plasmids pJJJl and P J J J ~kindly , ~ ~ provided by Dr J. Arrand (Manchester, UK), and subcloned into the BamHIIEcoRI and EcoRIIHindIII sites, respectively, of the transcription vector pBluescriptKS (Promega-Biotec, Madison, WI). For the preparation of IgLC RNA probes, the 550-bp SstI fragment containing the human IgLCK gene constant segment?6 and the 600-bp BglIII BumHI fragment containing the IgLCX gene C2 constant segm e ~ ~ respectively, t?~ were subcloned into pGEMl (Promega Biotec). Phages with the IgLCK and IgLCX genomic fragments were kindly provided by Dr P. Leder, (Cambridge, MA). After linearization with the appropriate restriction enzymes (GIBCO-BRL, Karlsruhe, Germany), [35S]-labeledantisense or sense (control) run-off transcripts were generated using either SP6, T3, or T7 RNA polymerasesand [35S]-uridine-5'-(u-thio)-triphosphate(1,250 Ci/mmol; New England Nuclear, Dreieich, Germany) or, altematively, digoxigenin-ll-uridine-5'-triphosphate(Boehringer Mannheim, Mannheim, Germany).38A mixture of EBER1- and EBER2specific RNA probes was applied to increase the sensitivity. In situ hybridization. The hybridization procedure with either [35S]-labeled,digoxigenin-labeled, or combinations of [35S]-and digoxigenin-labeledprobes on frozen and paran-sections, washing steps including RNase digestion of nonspecifically bound probe, and autoradiographywere performed as described.25Immobilized digoxigenin was detected using a monoclonal digoxigeninspecific antibody (Boehringer Mannheim; dilution 1:20) and the APAAP procedure. All sections were processed in parallel using the same batches of reagents and probes. Abrogation of specific signals in hybridizations preceded by micrococcal nuclease digest i ~ verified n ~ ~ that the targets of the hybridizations were RNA molecules. The specificity of the EBER probes was further tested on six HD-derived cell lines: of which only the line L591, known to harbor EBV,6expressed EBERl and EBER2. Sequential immunohistochemistryand in situ hybridization. Cryostat sections fixed in 4% paraformaldehyde were incubated in 2 mg/mL predigested pronase/lx phosphate-buffered saline (PBS) at 37°C for 5 minutes. Slides were rinsed twice with 0.1 mol/L glycine/lx Tris-buffered saline (TBS). Antibodies were used in 485 freshly prepared RPMI 1640 medium (GIBCO-BRL), pH 7.5, containing 10 mg/mL bovine serum albumin, 1.0 mg/mL yeast tRNA, and 5,000U/mL heparin ammonium salt (Sigma) to inhibit RNase activity.'IQ4*Incubation was 30 minutes at room temperature each with the monoclonal antibody, rabbit anti-mouse immunoglobulin antibody, and APAAP complex (Dakopatts), the latter two in 1:20 dilution, using RPMI dilution buffer as described above. The last two incubation steps were repeated once for 10 minutes each. Washing after each incubation step was done in lx TBS. Immobilized antibodies were visualized as described above and slides were immediately subjected to the in situ hybridization procedure. Paraffin sections were similarly treated following dewaxing in xylene and predigestion in 0.5 mg/mL protease VI11 (Sigma) in PBS for 8 minutes at room temperature; these further procedures did not differ from those for cryostat sections. Autoradiographic exposure was 4 to 6 days. To estimate the RNA loss during immunostaining procedures, adjacent tissue sections were subjected to in situ hybridization not preceded by immunostaining. The autoradiographic exposure times for slides subjected to the double-labelingprocedure were adjusted accordingly. RESULTS Histological diagnoses and phenotype characteristics of 46 HD cases are summarized in Table 1. Twenty-three cases showed EBER expression in neoplastic cells, while the other 23 cases did not display an EBER-specific signal in any of the H-RS cells even after prolonged autoradiographic exposure. The intensity of the EBER-specificin situ hybridization signal over H-RS cells varied considerably from cell to cell; short exposure times (6 to 12 hours of autoradiography) were particularly useful to assess the atypical nuclear morphologyof EBER+ H-RS cells, whereas extended exposure times ( > 4 days) resulted in exhaustion of the capacity of the autoradiographic emulsion over most EBER+ cells and were useful to establish the total number of such cells. The number of EBER+ cells was matched by the number of CD30+cells in adjacent tissue sections. Thus in these HD cases, virtually all H-RS cells, and not only a proportion, expressed EBER transcripts. Hybridization with sense (control) probes resulted in background signals over all cells. Eighteen of 23 cases with EBER+ H-RS cells displayed LMP expression in 10% to 80% of the neoplastic cells, as detected in paraffin sections. In comparison with the monoclonal antibodies, CS1-4, the LMP-specific rabbit antiserum stained smaller percentages of neoplastic cells. Unlike LMP expression, EBER transcripts were not restricted to H-RS cells. In most cases, small lymphoid cells with unsuspicious nuclear morphology were found in small Table 1. Phenotype of Hodgkin and Reed-SternbergCells in 46 HD Cases Diagnosis* EBER LMP CD30 CD20 CD3 HDlpn HDns HDmc HDld Total 2/2t 10124 212 8/24 212 24/24 212 2/24 Ioiia 7\18 miia 1/18 112 23/46 I I2 212 46/46 012 5/46 012 3/24 2/18 012 5/46 18/46 'Histotypes of HD: Ipn, lymphocyte predominance, nodular subtype; ns, nodular sclerosing: mc, mixed cellularity: Id, lymphocyte depletion. tNumber of cases with labeled H-RS cells per number of cases studied. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. HERBST ET AL 486 Table 2 Numben of EBER-Expmsslng Cells In 46 HD Cases With EBER* or EBER- H-RS Cells No. of EBER' c e l l s l 0 . k d Saction &ea 0 HD cases with EBER' H-RS cells HD cases with EBER- H-RS 2 cells AllHDcases 2 1-20 21-50 51-100 101-150 151-200 201.250 >250 - - - 2 1 1 2 11 5 5 1 1 - 2 - - 2 2 23 - 21 Elevated numbers of EBER' cells ( > 100) cells were found in 26 of 46 cases: in all those HD cases carrying EBER' H-RS cells (23 cases), as well as in three cases with EBER- tumor cells. numbers (Table 2). regardless of whether the neoplastic cells were EBV-infected (Fig 1). In two cases with EBER+ H-RS cells, no small EBER+ cells could be identified. In two additional biopsies, no EBER' cells were present at all, even in serial sections; three cases, two of which did not show EBV infection of the tumor cells, displayed high numbers of EBER-expressing small lymphoid cells, often accumulated in incoherent clusters. Two of the 10 control tissues with intact nodal architecture displayed only few interfollicular EBER+ cells (up to 10 per 0.5-cmZ section area); the other tissues did not show EBER-specific signals. Hybridizations with IgLC gene probes were performed with all tissues that did not contain EBER+ cells. These cases displayed intense labeling of plasma cells within 48 hours of exposure, thus excluding the possibility of a failure to detect EBER+ cells because of RNA degradation. Simultaneous isotopic/nonisotopic in situ hybridization double-labeling was used to characterize EBV-infected cells in each six of HDmc and HDns cases with LMP+ tumor cells and larger numbers of EBER+ small cells. With respect to EBER-specific labeling, the results were identical to those previously obtained with [35S]-labeledEBERspecific probes. In all cases, the tumor cells did not display any IgLC transcripts, whereas approximately each 30% of the small EBER+ cells showed IgLCK or IgLCA gene RNA, respectively (Fig 2). To further characterize EBER-expressing cells, nine HD cases, particularly cases with either an EBER- or EBER+/ CD20-/CD45RO-/TcRp- H-RS cell phenotype, were subjected to the sequential immunohistology/isotopic in situ hybridization double-labeling procedure on frozen and paraffin sections using antibodies against CD20, CD30, CD45R0, CD68, LMP, and the TcRp-chain. In cases with EBER+ H-RS cells, CD30-specific staining virtually matched with EBER expression (Fig 3, Table 3). LMP staining was always associated with EBER-specific autoradiographic signals, and CD68 reactivity was not coexpressed with EBER in any of the cases. Approximately 6 of 10 EBER+ reactive cells were detected by the CD20 antibody L26 in Flg 1. Dotodon of EBER hnraipts In th.cl.from HD p.tkntr. (A) EBER transcripts In H-RS cells In the absence of reactive EBER* cells; (B) multiple EBER+ small reactahre cells (dot-like labeling, small arrows) in the absence of EBER transcripts from H-AS (bold arrows); (C) absence of specific labeling using EBER-specific sense strand (control) probe In the same case as (A). Paraffin sections, in situ hybridization, PSIlabeled RNA antisense probes, 24 (A) and 48 (B,C) hours of autoradiographic exposure. (Original magnifications x410 AS; x 120 E.) From www.bloodjournal.org by guest on June 18, 2017. For personal use only. Fig 2. Detection of EBER (red substrate) and lgLCK gene transcripts (silver grains) by simultaneous in situ hybridization showing EBER+/lgLCK- H-RS cells, an EBER+/lgLCK+small B cell (arrow) with colocalization of both signals, and several EBER-/lgLCK+ small cells. Paraffin section, in situ hybridization with PsSI-labeled RNA antisense IgLoC probe and digoxigenin-labeled antisense EBER probes, APAAP procedure, autoradiographic exposure5 days, original magnification x540. Fig 3. Detection of EBERl and EBERZ transcripts in tissue from mixed cellularity type HD. in situ hybridization preceded by i"unostaining with the antibody Ber-HZ (CD30). EBER transcripts are almost exclusively associated with CD30+ atypical cells. The autoradiographic signal is heterogeneous, ranging from densely labeled cells t o cells with no or only few grains due t o the short exposure time of 24 hours. (Cryostat section; original magnification x410.) Fig 4. Detection of EBERl and EBERP transcripts in tissue from mixed cellularity type HD, in situ hybridization preceded by immunostaining with the antibody L26 (CDZO). EBER transcripts are associated with CD20- atypical cells and few CD20+ small cells (arrows). Cryostatsections, in situ hybridization, P?+labeled RNAantisenseprobes, autoradiographic exposure 24 hours. (Originalmagnification x290.1 Fig 5. In situ hybridization with EBER-specific antisense probes preceded by immunostaining with the antibody L26 (CD20). EBERspecific autoradiographic signal and immunostaining colocalized in a small lymphoid cell. H-RS cells are EBER- (arrow). HD, nodular sclerosing type, paraffin section, autoradiographic exposure 48 hours. (Original magnification x 250.) Fig 6. In situ hybridization with EBER-specific antisense probe preceded by immunostaining with the antibody L26 (CD20) showinga cluster of EBER+ reactive cells with weak or absent immunohistological signal. HD, mixed cellularity type, paraffin section, autoradiographic exposure 48 hours. (Original magnification x 120,) Fig 7. Detection of EBERl and EBERP transcripts in a mixed cellularity type HD case, in situ hybridization preceded by immunostaining with the antibody A6 (CD45RO). EBER transcripts in H-RS and reactive cells are absent from CD45RO+ cells. Paraffin section, in situ hybridization with [35S]-labeled RNA antisense probes, autoradiographic exposure 48 hours. (Original magnification x280.) From www.bloodjournal.org by guest on June 18, 2017. For personal use only. HERBST ET AL Table 3. Summary of Double-Labeling Results in a Case of Nodular SclerosingType HD With EBER+ H-RS Cells cells harbored EBV in H D cases with EBER+ H-RS cells. In frozen sections, CD30 staining identifies practically all H-RS cells in nearly all H D This observation is of Autoradiographic Exposure Time importance in the context of previous demonstrations of 24 h 96h monoclonality of EBV genomes in H D tissue extracts,5Y8-l0 EEER+/CD30+cells 90 96 and further supports the notion that infection of the EBER+/CD30- cells 2 2 neoplastic cells by EBV must have occurred before their EBER-/CD30+ cells 8 2 clonal expansion. The EBV infection is thus placed in an EEER+and/or CD30+ cells 100 100 appropriate time frame to have a role in the etiology of HD. Per 100 EEER+ and/or CD30+ cells; H-RS cells were identified by LMP expression in H-RS cells additionally supports this morphology and immunophenotype at the first time point, and at the concept because of the transforming potential of this gene second time point by CD3O-specific staining only, because the nuclear product in several experimental ~ystems.4~ LMP, which is morphologywas concealed by the autoradiographic signal. variably expressed during the course of the cell cycle,44 upregulates bcl-2 protein expression and, by this mechacryostat sections (Fig 4). In paraffin sections, the propornism, prevents programmed cell death in B cells.45 The tion of EBER+/CD20+ small cells was considerably lower presence of monoclonal EBV genomes and LMP expresand the staining was reduced to weak signal intensity, sion in H-RS cells is the most frequent abnormality with whereas neighboring EBER-/CD20+ cells were still strongly established oncogenic potential among H D cases and labeled (Figs 5 and 6). Furthermore, an HD case with supports the concept of an etiologic role of EBV in the CD20+/EBER+H-RS cells showed an intense CD20 stainpathogenesis of a significant proportion of H D cases. ing of the EBER-labeled tumor cells, further excluding a However, the presence of EBV in H-RS cells is restricted to mere technical problem resulting in reduced immunoreaca subset of HD, and certainly more than one immortalizing tivity of EBER+ small cells. Unambiguous reactivity of or transforming mechanisms are required for the generaEBER+ small cells with the TcRp-specific antibody PF1 or tion of malignant CD30+ cells. Therefore, more informathe antibody A6 (CD45RO) was not found in any of the tion, particularly on the interaction of EBV gene products tissues (Fig 7). EBER+ reactive cells were thus either of with cellular genes and proteins, is needed to conclusively B-cell phenotype (CD20+/IgLC+)or could not be assigned assess the significanceof the presently available data for the to a particular lineage. pathogenesis of HD and other EBV-associated malignanDISCUSSION cies. EBV was not restricted to neoplastic cells in biopsies Detection of the highly abundant EBER transcripts from HD patients. Variable, usually small numbers of represents a considerable technical advancement in the EBER+ reactive lymphoid cells were found in HD cases study of EBV infection, because it permits visualization of with EBER+ or EBER- H-RS cells. Absolute numbers practically all latently EBV infected cells in tissue sections. could be determined precisely in cases without EBV infecIn contrast to DNA-DNA in situ hybridization, use of tion of H-RS cells. In addition to the cytomorphologic single-stranded antisense EBER-specific RNA probes and aspect, sequential double-labeling using markers not exremoval of nonspecifically bound probe by ribonuclease pressed by the neoplastic cell population proved helpful to digestion result in low background signal and facilitate estimate the frequency of EBER+ reactive cells in tissues discrimination between EBV+ and EBV- ~ e l l s . 2It~ is - ~thus ~ with EBER+ H-RS cells. Some cases did not display any possible to address some of the questions that remained EBER+ cells even in serial sections. This absence of EBER open in previous studies, eg, as to the presence of EBV in expression did not represent a problem of tissue preservamainly neoplastic or reactive cells in HD tissue biopsies, and their number, distribution, and phenotype. tion, because IgLC gene transcripts could be detected in plasma cells and B cells. The presence of EBER+ reactive In situ hybridization for the detection of EBER transcripts showed the presence of EBV in H-RS cells of 23 of cells in only very small numbers, or even their absence, in most cases is likely to explain why EBER+ reactive cells 46 H D cases that were not included in any of our previous were not described in a previous report on EBER expresreports. Except for the two cases of HDlp, which may be not representative, HDmc was the histological type of the sion in HD.26 With respect to the normal lymph node tissues, similar results were obtained in a study focusing on disease most frequently associated with the virus, followed by HDns. These results are well in agreement with a nonmalignant lymphoid lesions.& The observation of recently published study describing EBER+ H-RS cells in EBER+ cells in only a low proportion of normal lymph nodes is in apparent contradiction to the fact that world11 of 23 cases of typical HD.” LMP was detectable by immunohistologyin variable proportions of EBER+ neoplaswide more than 90% of the population is seropositive for tic cells of 18 cases. This figure supports the view that EBV-specific antibodies. Quantitative evaluation of EBVprevious studies using EBV DNA in situ h y b r i d i z a t i ~ n ~ - ~ l Jinfected ~ peripheral blood cells, on the other hand, showed a or LMP i m m u n o h i ~ t o l o g yslightly ~ ~ ~ ~ underscored ~~~~ the very low frequency of EBV+ cells (1 to 5 infected cells per exact proportion of HD cases with EBV-harboring tumor lo7 mononuclear leukocytes) in healthy donors,” which is cells, whereas PCR studieW3 may have overestimated this in good agreement with the histomorphological findings. figure. Extension of the autoradiographic exposure time in Furthermore, antibody titers are unlikely to be determined combination with CD30-specific immunostaining verified only by EBV infection of lymphoid cells, because epithelial that virtually all, and not only a subset, of the neoplastic cells also represent targets for this herpes From www.bloodjournal.org by guest on June 18, 2017. For personal use only. EBV IN HODGKIN’S DISEASE 489 capsid a~~tigen,l~?~O and gp250/350 membrane antigen13) Counting of EBER+ cells per tissue section area showed from any cells in HD tissues. elevated numbers ( > 100) of such cells in all cases with EBER+ H-RS cells and, additionally, in three cases with In general, the majority of EBER+ cells were of (CD20+)/ EBER- neoplastic cells, ie, in 26 of 46 HD cases (56%; IgLC+/LMP- phenotype, whereas EBER+/TcRP+ or EBER+/CD45RO+ cells, ie, mature T cells, were not Table 2). This proportion is in good agreement with the This raises the possibility that immature lymresults of several semiquantitative PCR s t u d i e ~ ~ ~ J ~ detectable. J~J~ phoid cells, ie, cells with incomplete or missing antigen showing increased numbers (104 internal repetitive fragreceptor gene rearrangements cells, were among the ment target sequence^^^,^^) of EBV genomes in 55% to 60% EBER+/IgLC- subp~pulation.~~ However, our estimate of of the cases. The results presented here support the the proportions of EBER+ B and T cells is largely based on previous interpretation of PCR data that an increased EBV cases with elevated numbers of EBER+ reactive cells, genome content of HD tissues is most likely related to the presence of EBV in tumor cells, and in only rare instances which may not be representative for HD cases with small due to proliferations of EBV-infected reactive cells.1* numbers of EBER+ reactive cells. The presence of CD43+ Simultaneousdouble-labelingexperiments helped to charEBV-infected cells in addition to occasional B cells was acterize the majority of EBER+ reactive cells as IgLC+ recently observed in some cases of HD, and interpreted as mature B lymphocytes. The presence of both types of IgLC, evidence for a T-cell nature of these CD43+ cells.27However, CD43 antigen is not lineage-specific; moreover, it is K and A, indicates an oligoclonal or polyclonal composition found on many EBV+ lymphoblastoid B-cell lines.52Thus, of these cells; however, it does not provide information about the size of individual clones. H-RS cells did not whereas EBER+/TcRP+/CD45RO+small cells arising in certain pediatric immunodeficiencysyndromes can be idendisplay IgLC transcripts, which supports previous observatified with our methodology (H. Herbst, unpublished obsertion~:~suggesting their origin from immature B cells, T vation), it must be concluded that such EBER+ mature T cells, or other cells6 Immunophenotyping of reactive EBER+ cells was problematic and, particularly in paraffin cells, if present at all, appear to be extremely rare in lymph nodes from normal adults and HD patients. sections, virtually impossible for the vast majority of these The frequency of EBER+ cells in the reactive admixture cells. In frozen sections, most of the small EBER+ cells was slightly higher when compared with nonhyperplastic could be stained with the antibody L26 (CD20). However, control lymph nodes, but their phenotypic properties were in paraffin sections, the number of these cells was reduced. similar. The two groups of HD, with or without EBVCD20+/EBER- reactive cells and CD20+/EBER+ tumor harboring H-RS cells, respectively, did not differ from each cells, on the other hand, could be detected easily with our other with regard to the occurrence, frequency, and phenoimmunohistological procedure. These observations, in contype of reactive EBER+ cells. Thus, in the majority of HD junction with the IgLC+ phenotype of most EBER+ small patients, the postulated specific isolated immune defect cells and the established immunophenotype of resting and that permits proliferation of tumor cells expressing a viral activated, noninfected B lymphocytes as constantly CD20+ neo-antigen, LMP, is obviously not accompanied by an cells,@ led us to conclude that expression of the CD20 unrestricted proliferation of reactive EBER+ lymphoid antigen may be downregulated in latently EBV-infected cells. This is in sharp contrast to the pattern seen during small lymphoid cells in vivo. The CD20 antigen directly infectious mononucleosis,53 as well as in lymph nodes of regulates transmembrane ion flux in B lymphocytes and is a key structure for antigen-independent B-cell activati0n.4~ immunosuppressed transplant recipients or HIV-infected individuals,who generally display larger numbers of EBER+ Downregulation of this activation-associated antigen may cells.&It is thus likely that there are distinct immunological thus have specific advantages for EBV-harboring cells. To differences between patients with global immunodeficientest this hypothesis, it will be necessary to quantitate the cies, which are at risk to develop EBV-induced oligoclonal density of CD20 and other antigens on suspended cells, and or monoclonal non-Hodgkin’s lymph0ma,5~9~~ nonisotopic EBER-specific labeling may facilitate the idenon the one tification of EBV-infected cells in such experiments. LMP side, and HD patients on the other. expression was not observed in any of the reactive cells, ACKNOWLEDGMENT which is in keeping with published data,13.z1and may facilitate evasion of these cells from immune recognition. We thank H. Protz, T. Finn, and B. Nolte for technical This interpretation is also supported by the previously assistance, and L. Oehring for phototechnical assistance. Dr J. established absence of the EBNA2 latent gene prodArrand kindly supplied us with EBER plasmids, and Dr P. Leder generously provided phages with IgLC gene constant segments. U C ~ ~and ~ EBV , ~ ~lytic , ~cycle ~ antigens (early antigen,5O viral REFERENCES 4. Poppema S, van Imhoff G, Torensma R, Smit J: Lymphade1. Johansson B, Klein G, Henle W, Henle G: Epstein-Barr virus-associated antibody patterns in malignant lymphoma and nopathy morphologically consistent with Hodgkin’s disease associleukemia. Int J Cancer 6450,1970 ated with Epstein-Barr virus infection. Am J Clin Pathol 84:385, 1985 2. 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For personal use only. 1992 80: 484-491 Distribution and phenotype of Epstein-Barr virus-harboring cells in Hodgkin's disease H Herbst, E Steinbrecher, G Niedobitek, LS Young, L Brooks, N Muller-Lantzsch and H Stein Updated information and services can be found at: http://www.bloodjournal.org/content/80/2/484.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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